β-mannanase having improved production yields and enzymatic activity

ABSTRACT

A β-mannanase having increased production yield and enzymatic activity is disclosed. The β-mannanase has a modified amino acid sequence of SEQ ID NO: 2, wherein the modification is a substitution of Tyrosine at position 25 with Histidine.

FIELD OF THE INVENTION

The present invention relates to a β-mannanase, and more particularly toa β-mannanase having improved production yield and enzymatic activity.

BACKGROUND OF THE INVENTION

β-1,4 Mannans are major components of hemicellulose in plant cell wallof softwood, plant seeds and beans. Four types of polysaccharidesincluding linear mannan, galactomannan, glucomannan, galactoglucomannanthat are linked via β-1,4-glycosidic bonds compose mannans. Mannanhydrolysis provides wide array of biotechnological applications, such asfeed manufacture, pulp and paper industries, and hydrolyzing coffeeextract to reduce viscosity. A set of enzymes are required for completedegradation of mannans, including endo-β-1,4-mannanase (β-mannanase, EC3.2.1.78), exo-β-mannosidase (EC 3.2.1.25) to cleave the main chain, andβ-glucosidase (EC 3.2.1.21), α-galactosidase (EC 3.2.1.22), and acetylmannan esterase to remove side chain decoration. Among them, β-mannanasewhich catalyzes random hydrolysis of manno-glycosidic bonds in the mainchain is the key enzyme. More recently, major products of β-mannanase,mannotriose and mannobiose (mannooligosaccharides, MOS), have beenproved beneficial as animal nutrition additive due to its prebioticproperties.

β-Mannanases are derived from various organisms including bacteria,yeasts, and filamentous fungi. According to the amino acid sequencehomology, β-mannanases are mostly classified to glycoside hydrolase (GH)families 5, 26 and 113. These families share the same (β/α)₈ folding andcatalytic machinery, that two glutamate residues at active site serve asgeneral acid/base and nucleophile to catalyze the cleavage of glycosidicbonds via a retaining double displacement mechanisms. Since industrialprocess is usually carried out at high temperatures, stable enzyme usageunder a broad range of temperature is highly desirable. Therefore,β-mannanase needs to be modified to meet the requirement for differentindustrial usages. There are two ways to achieve these goals, one way isto screen suitable genes in nature, and the second way is modifyingcurrent enzyme genes based on their 3-D structural information.

In the present invention, the crystal structure of β-mannanase isanalyzed and the enzyme activity of β-mannanase is improved bysite-directed mutagenesis of the gene.

SUMMARY OF THE INVENTION

An object of the present invention is to modify β-mannanase by means ofstructural analysis and site-directed mutagenesis to efficientlyincrease the production yield and the enzymatic activity, and improveits economic value of industrial application.

According to an aspect of the present invention, there is provided aβ-mannanase having increased enzymatic activity. The β-mannanase has amodified amino acid sequence of SEQ ID NO: 2, wherein the modificationis a substitution of Tyrosine at position 25 with Histidine.

In an embodiment, the amino acid sequence of SEQ ID NO: 2 is encoded byManBK gene isolated from Aspergillus niger BK01, and the β-mannanase isan acidic and thermotolerant mannanase.

In an embodiment, the β-mannanase has a full length amino acid sequenceof SEQ ID NO: 4.

In an embodiment, the β-mannanase is used in a food industry, a feedindustry, or a paper pulp industry.

The above objects and advantages of the present invention will becomemore readily apparent to those ordinarily skilled in the art afterreviewing the following detailed description and accompanying drawings,in which:

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the gene sequence and the amino acid sequence of thewild-type ManBK;

FIG. 2 shows the protein structure of the wild-type ManBK, which wassuperimposed with Trichoderma reesei mannanase in complex withmannobiose;

FIG. 3 shows the sequence of the mutagenic primer for the Y25H mutant;

FIG. 4 shows the gene sequence and the amino acid sequence of the Y25Hmutant;

FIG. 5 shows the production yields and the activity analyses of thewild-type ManBK and the Y25H mutant; and

FIG. 6 shows the thermostability analyses of the wild-type ManBK and theY25H mutant.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention will now be described more specifically withreference to the following embodiments. It is to be noted that thefollowing descriptions of preferred embodiments of this invention arepresented herein for purpose of illustration and description only; it isnot intended to be exhaustive or to be limited to the precise formdisclosed.

In the present invention, a gene of the β-mannanase ManBK was isolatedfrom Aspergillus niger BK01, and ManBK is an acidic and thermotolerantβ-mannanase. In order to improve the industrial application value ofthis enzyme, the protein structure of the apo-form ManBK was solved byX-ray crystallography, and the solved structure was superimposed withTrichoderma reesei mannanase (having 57% similarity in protein sequencecompared with ManBK) in complex with mannobiose. Then, based on thestructural information of the enzyme, the important amino acid residuesin the active site were selected for site-directed mutagenesis toimprove the enzymatic activity. The enzyme modification process of ManBKand the resulted mannanase protein are described in detail as follows.

First, the ManBK gene was obtained from Aspergillus niger BK01 (GenBankaccession no. FJ268574), and as shown in FIG. 1, the full length ofsequence of the ManBK gene is 1038 base pairs (SEQ ID NO: 1), whichencodes a protein of 345 amino acids (SEQ ID NO: 2). The ManBK gene wasconstructed into pPICZαA vector by using EcoRI and NotI sites. Theprimers for polymerase chain reaction were 5′-GGTATTGAGGGTCGCGCGGCGGCGGCGGCGATGTCCTTCGCTTCCACTTCCG-3′ (SEQ ID NO: 5, forward primer) and5′-AGAGGAGAGTTAGAGCCTTAAGCGGAACCGATAGCA GC-3′ (SEQ ID NO: 6, reverseprimer). The constructed plasmid was transformed into a competent cellas a wild-type expression vector.

To solve the protein structure of ManBK by X-ray crystallography, theprotein crystal was obtained by using sitting drop vapor diffusionmethod at room temperature by Hampton screen kit. The protein crystal ofManBK in apo form was prepared by mixing 2 μl mannanase solution (10mg/ml in 25 mM Tris-HCl, pH 7.5) with equal amounts of mixture solutionand mother liquor, and equilibrating with 500 μl of the mother liquor atroom temperature. The wild-type ManBK crystal was obtained by acondition composed of 0.1M Bis-Tris pH 5.5, 0.4M magnesium chloride, and29% PEG3350. The molecular replacement method was used for phasing X-raydiffraction data, and the protein structure of ManBK was subsequentlydetermined by crystallographic software.

FIG. 2 shows the protein structure of ManBK solved by X-raycrystallography, and the solved structure was superimposed withTrichoderma reesei mannanase in complex with mannobiose in subsites +1and +2. The protein structure of ManBK has (β/α)₈ barrel fold, wherein 8β-sheets are located in the interior and 8 α-helixes pack around theexterior. By studying the structural information of ManBK, 30 amino acidresidues were selected to be modified. Particularly, Tyr25 is located inthe active site of the enzyme and may be important to the catalyticreaction of ManBK, and thus is targeted for site-directed mutagenesis,and it is found that the mutation of Tyr25 improves the production yieldand the enzymatic activity of ManBK, while other mutations do not showsignificant effects and are not redundantly described here. Thefollowing describes the processes for site-directed mutagenesis, proteinexpression and activity assay of Y25H mutant.

The Y25H mutant was prepared by using QuikChange site-directedmutagenesis kit with ManBK gene as a template. The sequence (SEQ ID NO:7) of the primer for Y25H mutant was shown in FIG. 3, wherein Y25H meansTyrosine at position 25 was mutated into Histidine; in other words, themodification is a substitution of Tyrosine at position 25 withHistidine. The original template was removed via DpnI digestion under37° C., and then the plasmid with mutated gene was transformed into E.coli and screened with Ampicillin. Finally, the mutated gene wasconfirmed by DNA sequencing. Therefore, the Y25H mutant was constructed,and as shown in FIG. 4, the gene sequence was numbered as SEQ ID NO: 3,and the amino acid sequence was numbered as SEQ ID NO: 4.

The wild-type and mutant ManBK were expressed in Pichia. First, theplasmid DNA was linearized by PmeI and transformed into the P. pastorisX33 strain by electroporation. The transformants were selected on YPD(1% yeast extract, 2% peptone, 2% glucose, 2% agar) plates containing100 μg/mL Zeocin and incubated at 30° C. for 2 days. The picked colonieswere inoculated into 5 ml YPD medium at 30° C. overnight and furtheramplified into 50 ml BMGY medium at 30° C. overnight. After that, thecultured medium was changed to 20 ml BMMY with 0.5% methanol to inducethe target protein expression. The samples were collected at differenttime points for every 24 hours, and meanwhile, the methanol was addedinto the flask to the final concentration of 0.5%. After induction for 4days, the cells were harvested by centrifugation at 3500 rpm and thesupernatant was collected for further purification.

The supernatant was purified by FPLC (fast protein liquidchromatography) using Ni²⁺ column and DEAE column. Finally, thewild-type and mutant ManBK proteins, which had above 95% purity, wereconcentrated up to 5 mg/ml in protein buffer (25 mM Tris and 150 mMNaCl, pH 7.5) and then stored at −80° C.

To verify the difference between the wild-type and mutant ManBK, theproduction yields thereof were measured, and the β-mannanase activityassay and the thermostability analysis were performed. The β-mannanaseactivity was determined by dinitrosalicylic acid (DNS) method usingmannose as a standard. The reaction was started by mixing 0.2 mLappropriately diluted enzyme sample with 1.8 mL of 3 mg/L locust beangum (LBG) in 0.05 M citrate acid, pH 5.3. After 5-min incubation at 50°C., the reaction was stopped by adding 3 ml of DNS-reagent and boiledfor 5 min to remove residual enzyme activity. After cooling in coldwater bath for 5 min, the 540 nm absorbance of the reaction solution wasmeasured. One unit of β-mannanase activity was defined as the amount ofenzyme releasing 1 μmol of mannose equivalents per minute per mg oftotal soluble proteins under the assay conditions.

FIG. 5 shows the production yields and the activity analyses of thewild-type ManBK and the Y25H mutant. The genes of the wild-type ManBKand the Y25H mutant were transformed into Pichia for expression, and theextracellular production yields and the β-mannanase activities weremeasured. The results showed that the production yield of the Y25Hmutant was significantly higher than that of the wild-type ManBK and hada 1.68-fold increase. Moreover, the β-mannanase activity of the Y25Hmutant was also significantly higher than that of the wild-type ManBKand had a 1.5-fold increase.

For thermo stability analysis, the wild-type ManBK and the Y25H mutanthaving the same protein concentration were heat-treated at severaltemperature points from 50° C. to 90° C. with an interval of 5° C. for 2min, and then incubated on ice for 5 min. Afterwards, the enzymaticactivities were measured as mentioned above.

FIG. 6 shows the thermostability analyses of the wild-type ManBK and theY25H mutant. The results showed that there was no significant differencefor thermostability between the wild-type ManBK and the Y25H mutant. Forexample, after the heat treatment of 80° C., both the wild-type ManBKand the Y25H mutant maintained 80% activities when compared with theiroriginal activities. That is to say, the mutation to substitute Tyrosineat position 25 with Histidine did not affect the thermostability of theenzyme.

In addition, when the wild-type ManBK and the Y25H mutant weretransformed into Pichia and expressed in a 50 L fermentor, theproduction yield and the β-mannanase activity of the Y25H mutant wereboth significantly higher than those of the wild-type ManBK, and thetotal activity of the Y25H mutant had a 1.5-fold increase when comparedwith the wild-type ManBK. In other words, the production yield and theβ-mannanase activity of the Y25H mutant were both significantly higherthan those of the wild-type ManBK no matter by using flask or 50 Lfermentor test. Therefore, for the industries in need of thethermostable mannanase, the Y25H mutant having improved production yieldand enzymatic activity can reduce the production cost and increase itscompetitiveness in the industries.

From the above, in order to improve the production yield and theenzymatic activity of ManBK, the present invention solved the proteinstructure of the apo-form ManBK by X-ray crystallography, and the ManBKstructure was superimposed with Trichoderma reesei mannanase complexstructure. According to the superimposed structure, Tyr25 which islocated in the active site is selected for site-directed mutagenesis andthe Tyrosine at position 25 was mutated into Histidine to construct theY25H mutant. The Y25H mutant exhibited significantly increasedproduction yield and enzymatic activity when compared with thewild-type, and the thermostability of the enzyme is not affected, so itcan reduce the production cost and will has more industrialapplications. In addition, since ManBK has thermostability and can beapplied to many industries with thermal processes, once the productionyield and the enzymatic activity thereof is increased, the productioncost will be reduced and the profit will be increased. Therefore, thepresent invention successfully modified ManBK to improve the productionyield and the enzymatic activity thereof, and thus, the presentinvention possesses high industrial value.

While the invention has been described in terms of what is presentlyconsidered to be the most practical and preferred embodiments, it is tobe understood that the invention needs not be limited to the disclosedembodiment. On the contrary, it is intended to cover variousmodifications and similar arrangements included within the spirit andscope of the appended claims which are to be accorded with the broadestinterpretation so as to encompass all such modifications and similarstructures.

What is claimed is:
 1. A β-mannanase comprising a modified amino acidsequence of SEQ ID NO: 2, wherein the modification is a substitution ofTyrosine at position 25 with Histidine.
 2. The β-mannanase according toclaim 1 wherein the amino acid sequence of SEQ ID NO: 2 is encoded byManBK gene isolated from Aspergillus niger BK01.
 3. The β-mannanaseaccording to claim 1 being an acidic and thermotolerant mannanase. 4.The β-mannanase according to claim 1 having a full length amino acidsequence of SEQ ID NO:
 4. 5. The β-mannanase according to claim 1wherein the β-mannanase is used in a food industry, a feed industry, ora paper pulp industry.